Mammalian breathing is a vital behavior whose underlying neural mechanism originates in the brainstem. This project aims to determine the components of the brainstem neural circuits that generate and control respiration in mammals, including humans, and thus has significant implications for human health. A site in the ventral medulla called preBotzinger Complex (preBotC) is essential for breathing. However, the cellular composition of the preBotC, in terms of the genetic and developmental identity of the different cell populations it contains, and their physiological properties, remain largely unknown. A genetically distinct subpopulation of neurons in the preBotC is hypothesized to form the kernel that generates respiratory rhythm. To evaluate the role of these key neurons, a multidisciplinary research approach is employed that combines molecular genetics and electrophysiology. Recently developed technologies that deliver cell lineage markers via genetic methods, such as site-specific recombination and fluorescent tagging, have greatly impacted neural development studies. Transgenic knock-in mouse models engineered with recombinase-fused cell lineage tracers and reporter transgenes are essential components of the research project. Likewise, characterization of these genetically distinct neuronal populations is carried out through electrophysiological recordings using a unique in vitro brainstem slice preparation that contains essential respiratory neural circuits and allows both cellular-level and systems-level recordings of respiratory motor output. Thus, this research project can evaluate the importance of the key population of neurons in breathing with a multilevel approach: molecular, cellular and system-level properties will be analyzed. Specific Aim 1 will assess the rhythmogenic role of these neurons through reversible genetic silencing and irreversible laser lesioning. Specific Aim 2 will evaluate the membrane properties of the key neurons consistent with their rhythmogenic role through whole-cell recordings. This project will elucidate the neural origins of mammalian respiration. The new knowledge obtained in this project will advance our understanding in the diagnosis and treatment of respiratory disorders that result from dysfunctions in the central nervous system, and provide key new knowledge regarding rhythm generation, which is generally applicable to understanding brain function. PUBLIC HEALTH RELEVANCE: Breathing is a human behavior that is essential in maintaining life. This project aims to reveal the cellular composition of brainstem neural circuits that generate and control breathing rhythms, and to characterize the properties of these cells consistent with their role as rhythm generators. The new knowledge acquired will facilitate the diagnosis and treatment of respiratory disorders with a central neural etiology, and elucidate the neural mechanisms that underlie rhythmic motor behaviors in general.